RESUMEN
Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40 MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system's performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.
Asunto(s)
Microscopía/instrumentación , Microscopía/métodos , Algoritmos , Calibración , Línea Celular Tumoral , Colorantes/química , Fluorescencia , Análisis de Fourier , Humanos , Soluciones , Factores de TiempoRESUMEN
The ability to quantify the fluorescence signals from multiply labeled biological samples is highly desirable in the life sciences but often difficult, because of spectral overlap between fluorescent species and the presence of autofluorescence. Several so called unmixing algorithms have been developed to address this problem. Here, we present a novel algorithm that combines measurements of lifetime and spectrum to achieve unmixing without a priori information on the spectral properties of the fluorophore labels. The only assumption made is that the lifetimes of the fluorophores differ. Our method combines global analysis for a measurement of lifetime distributions with singular value decomposition to recover individual fluorescence spectra. We demonstrate the technique on simulated datasets and subsequently by an experiment on a biological sample. The method is computationally efficient and straightforward to implement. Applications range from histopathology of complex and multiply labelled samples to functional imaging in live cells.
Asunto(s)
Algoritmos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Inteligencia Artificial , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Health Informatics World Wide, a Web index of research and educational institutions in the fields of Health Informatics and Medical Informatics has been available to WWW users since 1995. In this paper we report on the original conception of this service, its subsequent modifications and address its maintenance and related problems. Access statistics are presented which demonstrate its relevance as well as the geographical focus of Health Informatics research and development.
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Indización y Redacción de Resúmenes , Internet , Informática Médica , HipermediaRESUMEN
A series of pyrimidine thioethers was synthesized and evaluated for inhibitory properties against wild-type HIV-1 reverse transcriptase (RT) and an RT carrying the resistance-conferring mutation P236L. Modifications of both the pyrimidine and the functionality attached through the thioether yielded several analogues, which demonstrated activity against both enzyme types, with IC50 values as low as 190 nM against wild-type and 66 nM against P236L RT. Evaluation of a select number of pyrimidine thioethers in cell culture showed that these compounds have excellent activity against HIV-1IIIB-WT and retain good activity against a laboratory-derived HIV-1MF delavirdine-resistant variant.
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Fármacos Anti-VIH , Delavirdina/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Pirimidinas , Inhibidores de la Transcriptasa Inversa , Inhibidores de la Transcriptasa Inversa/síntesis química , Sulfuros , Sustitución de Aminoácidos , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Leucina/genética , Ratones , Prolina/genética , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Sulfuros/síntesis química , Sulfuros/química , Sulfuros/farmacologíaAsunto(s)
Benzopiranos/farmacología , Trastornos Migrañosos/tratamiento farmacológico , Piperazinas/farmacología , Receptores de Serotonina/efectos de los fármacos , Agonistas de Receptores de Serotonina/farmacología , Vasoconstrictores/farmacología , Animales , Benzopiranos/química , Benzopiranos/metabolismo , Benzopiranos/toxicidad , Disponibilidad Biológica , Presión Sanguínea/efectos de los fármacos , Encéfalo/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/fisiología , Gatos , Línea Celular , Evaluación Preclínica de Medicamentos , Duramadre/irrigación sanguínea , Duramadre/efectos de los fármacos , Gorilla gorilla , Cobayas , Hipotermia/metabolismo , Inflamación/fisiopatología , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/toxicidad , Receptor de Serotonina 5-HT1D , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/toxicidad , Estereoisomerismo , Sumatriptán/metabolismo , Sumatriptán/farmacología , Sumatriptán/toxicidad , Resistencia Vascular/efectos de los fármacos , Vasoconstrictores/química , Vasoconstrictores/metabolismo , Vasoconstrictores/toxicidadAsunto(s)
Fármacos Anti-VIH/farmacología , VIH-1 , Piridinas/farmacología , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacocinética , Línea Celular , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Masculino , Mutación , Piridinas/síntesis química , Piridinas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacocinética , EstereoisomerismoRESUMEN
Bisphosphonate ester 2 is an inhibitor of inflammation, but is devoid of antiarthritic effects. SAR studies on a series of related bisphosphonate esters resulted in compounds 6e, 6i, 6j, and 6m, which exhibited excellent inhibition of an arthritis model, in addition to potent anti-inflammatory effects.
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Antiinflamatorios no Esteroideos/síntesis química , Artritis Experimental/tratamiento farmacológico , Difosfonatos/síntesis química , Inflamación/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/uso terapéutico , Difosfonatos/química , Difosfonatos/uso terapéutico , Diseño de Fármacos , Granuloma/tratamiento farmacológico , Granuloma/inmunología , Hipersensibilidad Tardía , Indicadores y Reactivos , Ratones , Estructura Molecular , Relación Estructura-ActividadAsunto(s)
Nutrición Enteral/instrumentación , Esófago , Gastrostomía/instrumentación , Intubación Gastrointestinal/instrumentación , Stents , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/terapia , Esofagoscopios , Migración de Cuerpo Extraño/prevención & control , Humanos , SuturasRESUMEN
We have discovered two substituted 4-aminopiperidine compounds having high in vitro affinity and selectivity for the human dopamine D1 receptor. Both compounds, 3-ethoxy-N-methyl-N-[1-(phenylmethyl)-4-piperidinyl]-2-pyridinylamine (U-99363E), and its 3-isopropoxy analog (U-101958), were found through a routine receptor binding screen. The determined affinities (Ki) of these compounds for the cloned human dopamine D4 receptor were 2.2 and 1.4 nM, respectively. They exhibited at least 100-fold lower affinities for dopamine D2 and for other dopaminergic, serotonergic and adrenergic receptors. Both compounds were found to antagonize quinpirole-induced mitogenesis in Chinese hamster ovary cells expressing the human dopamine D4 receptor. In spite of their poor metabolic stability and low bioavailability. U-99363E and U-101958 appear to be among the first high-affinity, highly selective dopamine D4 receptor antagonists reported, and may have utility in in vitro investigations requiring selective tagging or blockade of dopamine D4 sites.
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Aminopiridinas/farmacología , Piperidinas/farmacología , Receptores de Dopamina D2/efectos de los fármacos , Aminopiridinas/metabolismo , Animales , Células CHO , Cricetinae , Humanos , Mitosis/efectos de los fármacos , Piperidinas/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4 , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transducción de SeñalAsunto(s)
Antipsicóticos/síntesis química , Antagonistas de Dopamina/síntesis química , Antagonistas de los Receptores de Dopamina D2 , Piperazinas/síntesis química , Sulfonamidas/síntesis química , Animales , Antipsicóticos/química , Antipsicóticos/metabolismo , Antipsicóticos/farmacología , Disponibilidad Biológica , Células CHO , Cricetinae , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/química , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Ergolinas/antagonistas & inhibidores , Humanos , Estructura Molecular , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Unión Proteica , Quinpirol , Receptores Adrenérgicos/metabolismo , Receptores de Dopamina D4 , Receptores de Serotonina/metabolismo , Esquizofrenia/tratamiento farmacológico , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologíaRESUMEN
A study of the decomposition of the pyrazoline bisphosphonate ester 2 identified 3 as the sole bisphosphonate component. Evaluation in a delayed-type hypersensitivity granuloma model of chronic inflammation in mice (DTH-GRA) showed 3 to be a potent inhibitor of granuloma formation (sc, 10 mg/kg, 45%), but in a murine model of antigen-induced arthritis (AIA), no significant inhibition was observed. As a result, new ketonic bisphosphonate tetraethyl esters were synthesized from vinylidenebisphosphonic acid tetraethyl ester 4 and activated carbonyl compounds in 13-84% yield. 6 significantly inhibited the pathology of both the DTH-GRA (sc, 25 mg/kg, 45%) and AIA models (sc, 25 mg/kg, 55%). Other compounds in the series were not as potent. Our results show that bisphosphonate ester 6 can inhibit the chronic inflammatory response associated with cutaneous granuloma formation and erosive arthritis.
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Antiinflamatorios no Esteroideos/síntesis química , Artritis/tratamiento farmacológico , Difosfonatos/síntesis química , Animales , Antiinflamatorios no Esteroideos/farmacología , Difosfonatos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Ratones , Ratones Endogámicos C57BL , Relación Estructura-ActividadRESUMEN
Dopamine receptors play an important role in neurotransmission within the central nervous system, as evidenced by their affinity to and apparent site of action of a variety of psychotropic agents. The cloning of dopamine receptor subtypes makes possible the discovery of subtype-selective agonist and antagonist compounds useful for the study of receptor physiology and for the development of more effective treatments for disturbances stemming from aberrant dopamine neurotransmission. We describe in this report the receptor pharmacology of a human D4-dopamine receptor expressed in HEK293 cells from a synthetic gene. The affinities of monoaminergic antagonists in competition with [3H]spiperone-labeled sites in these cells matched very closely their rank order potency measured by other investigators for D4 receptors expressed in COS-7 cells. Further, the sites expressed in the HEK293-D4-24 line met six specific pharmacological criteria by which the D4 subtype has been distinguished from other D2-like dopamine receptors. The site expressed in this cell investigators for D4 receptors expressed in COS-7 cells. Further, the sites expressed in the HEK293-D4-24 line met six specific pharmacological criteria by which the D4 subtype has been distinguished from other D2-like dopamine receptors. The site expressed in this cell line thus bears the earmarks of a human D4-dopamine receptor. Competition of several agonists best fit a two-site binding model. This result, along with the measured 10-fold GTP-shift for dopamine, strongly suggests functional coupling of this receptor to endogenous G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Riñón/ultraestructura , Receptores de Dopamina D2 , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Animales , Unión Competitiva , Células CHO/metabolismo , Células CHO/ultraestructura , Células Cultivadas , Cricetinae , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Embrión de Mamíferos , Humanos , Riñón/química , Receptores Dopaminérgicos/genética , Receptores de Dopamina D4 , Sensibilidad y EspecificidadRESUMEN
Diphosphonates (DP) are synthetic pyrophosphates with a P-C-P backbone and are predominantly used for the treatment of bone diseases. Several DP have also been shown to exert significant antiarthritic effects in the rat adjuvant-induced polyarthritis model; however, there is no direct evidence for the anti-inflammatory effects of these compounds. We therefore tested the effects of dichloromethylene diphosphonate on delayed-type hypersensitivity granuloma elicited by s.c. implantation of antigen-soaked hydroxyapatite disks in antigen-sensitized mice. Dichloromethylene diphosphonate induced a dose-related inhibition of the delayed-type hypersensitivity granuloma response (38-64% at 25-100 mg/kg/day s.c. or p.o.); novel DP analogs, U-81581, U-82579 and U-84849 were also effective in the same dose range. In contrast, all DP failed to suppress 24-hr delayed-type hypersensitivity paw edema in mice. In addition to rat adjuvant-induced polyarthritis, mouse antigen-induced erosive arthritis was also significantly suppressed by s.c. administration of all four DP. Toxicity was minimal for each DP (> 600 mg/kg p.o. or s.c.). We conclude that DP represent a novel class of anti-inflammatory agents with excellent therapeutic potential for chronic inflammatory diseases including rheumatoid arthritis.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Difosfonatos/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Edema/tratamiento farmacológico , Femenino , Granuloma de Cuerpo Extraño/tratamiento farmacológico , Hipersensibilidad Tardía/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratas , Ratas WistarRESUMEN
Vinylidenebisphosphonic acid tetraethyl ester (1) and diazo ketones 7a-1 in ether at 22 degrees C yield pyrazoline bisphosphonate tetraethyl esters 8a-1 in moderate to good yield. These compounds were evaluated in animal models of arthritis: rat adjuvant-induced polyarthritis (AIP) and murine antigen-induced arthritis (AIA) and a murine model of chronic inflammation, the delayed type hypersensitivity granuloma reaction (DTH-GRA). (5-Benzoyl-2,4-dihydro-3H-pyrazol-3-ylidene)-bisphosphonic acid tetraethyl ester (8a), and [5-(3-fluorobenzoyl)-2,4-dihydro-3H-pyraxol-3-ylidene]- bisphosphonic acid tetraethyl ester (8d) significantly inhibited the arthritis models, AIP (15 mg/kg) and AIA (25 mg/kg), as well as the DTH-GRA (25 mg/kg). Conversion of 8a to the corresponding bisphosphonic acid, 10a, resulted in loss of activity. Compounds with alkyl substituents on the pyrazoline nitrogen, 9a-d, were inactive in the DTH-GRA. These results show that 8a and 8d have novel antiinflammatory activity and are capable of inhibiting chronic arthritis and inflammation in animals. Such compounds might be useful in man for treating chronic tissue injury associated with arthropathies such as inflammatory joint disease as well as other chronic inflammatory diseases.
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Antiinflamatorios no Esteroideos/síntesis química , Difosfonatos/síntesis química , Pirazoles/síntesis química , Animales , Artritis/tratamiento farmacológico , Difosfonatos/química , Difosfonatos/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Pirazoles/química , Pirazoles/farmacología , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
In 1974 the University of Kentucky was faced with two problems: 1) overburdening of local clinical facilities by students and 2) a large exodus of graduates from the state. The advent of the Area Health Education System offered the opportunity for the clinical education program to develop nontraditional clinical sites across the state. The development and use of these nontraditional sites in predominantly rural areas has become an integral part of the clinical education program. Local facilities are no longer inundated with students. The retention rate of graduates has improved in the seven years of the program from 36 percent to 1972 to 81 percent in 1979.